Method for increasing survival rate of cells in animal cell culture under hypoxia condition

ABSTRACT

The present invention relates to a method for increasing survival rate of cells in animal cell culture under hypoxia condition by adding antibiotics to the culture media. The method of present invention comprises a step of culturing animal cells in culture media containing antibacterial agent of quinolones, quinones, aminoglycosides or chloramphenicol at the concentration range of 0.1 to 1000 μg/ml. The invented method can be practically applied for high-density animal cell culture to produce recombinant proteins or cultured cells.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a method for increasingviability of animal cells in culture under hypoxia condition, morespecifically, to a method for increasing survival rate of cells inanimal culture under hypoxia condition by adding antibiotics to theculture medium.

[0003] 2. Description of the Prior Art

[0004] Most of animal cells require oxygen as a substrate in addition tonutrients for living. Thus, insufficient supply of oxygen to cells maycause various problems in medicine and industry. For example, indeveloping artificial organs including artificial liver, if the supplyof nutrients and oxygen is hampered by limitation of mass transfer intothe cells, the cells become died, especially, in case of usingencapsulated cells, oxygen transfer is a more serióus problem (see:Catapano et al., Int. J. Artif. Organs, 19(1):61-71, 1996).

[0005] In case of myocardial infarction and cerebral infarction, theblockage of blood vessels by which oxygen is supplied to tissues mayhinder blood flow, resulting in necrosis of the tissues (see: Selwyn etal., Ischemic heart disease, 1077-1085, In: Isselbacher et al. (eds.),Harrison's Principles of Internal Medicine, 13th ed., McGraw-Hill, Inc.,New York). In this case, an inadequate supply of glucose which is usedas an energy source by cells as well as an inadequate supply of oxygenmake the situation more serious.

[0006] Recently, high-density animal cell culture is one of the populartechniques used for production of recombinant proteins or for productionof cultured cells. As animal cells do not have cell walls differentlyfrom microorganisms, animal cell membranes may be easily destroyed bymechanical agitation or a contact with air, which makes it verydifficult to supply oxygen into culture medium by agitation, resultingin reduction of final concentration of the cells.

[0007] In order to solve the oxygen transfer problem, attempted are amethod for increasing dissolved oxygen concentration by adding purifiedhemoglobin which can bind to perfluorohydrocarbon or oxygen; a methodfor increasing yield of energy production using electron acceptors suchas is fumaric acid other than oxygen; and, a method for increasingavailable oxygen inside the cells by expressing genetically manipulatedhemoglobin in the cells. Also, recently attempted is a method forincreasing resistance of cardiac cells to hypoxic condition by affectingenergy metabolism pathway using trimetazidine. The said methods,however, have revealed disadvantages as followings: first, there is alimitation in improving the efficacy by adding perfluorohydrocarbon orhemoglobin to a culture medium since it does not change intrinsicproperty of cells but simply increases concentration of dissolved oxygenor promotes oxygen transfer; secondly, there is a limitation in aneffective concentration range of electron acceptors like fumaric acidsince the electron acceptors become reduced; thirdly, the method forincreasing oxygen transfer by expressing recombinant genes in the cellsrequires complicated process and is very costly.

[0008] Under the circumstances, there are strong reasons for exploringand developing an alternative method for increasing viability of animalcells in culture under a low oxygen condition.

SUMMARY OF THE INVENTION

[0009] The present inventors have made an effort to develop a method forincreasing the viability of animal cells in culture under hypoxiacondition, and found that the survival rate of animal cells in cultureunder a low oxygen condition can be dramatically increased by growingcells in a medium containing antibiotics of quinolones, quinones,aminoglycosides or chloramohenicol in a concentration range of 0.1-1000μg/ml.

[0010] The primary object of the present invention is, therefore, toprovide a method for increasing survival rate of cells in animal cellculture under hypoxia condition.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The above and the other objects and features of the presentinvention will become apparent from the following descriptions given inconjunction with the accompanying drawings, in which:

[0012]FIG. 1 is a graph showing HepG2 cell viability under variousoxygen conditions.

[0013]FIG. 2a is a graph showing cell viability of HepG2 with incubationtime under a low oxygen and a low glucose condition.

[0014]FIG. 2b is a graph showing the change in residual glucoseconcentration with incubation time under a low oxygen and a low glucosecondition.

[0015]FIG. 2c a graph showing the change in pH with incubation timeunder a low oxygen and a low glucose condition.

[0016]FIG. 3a is a graph snowing cell viability of HepG2 with incubationtime under a low oxygen and a high glucose condition.

[0017]FIG. 3b is a graph showing the change in residual glucoseconcentration with incubation time under a low oxygen and a low glucosecondition.

[0018]FIG. 3c is a graph showing the change in pH with incubation timeunder a low oxygen and a low glucose condition.

[0019]FIG. 4a is a graph showing cell viability of HepG2 with incubationtime under a normal oxygen and a low glucose condition.

[0020]FIG. 4b is a graph showing the change in residual glucoseconcentration with incubation time under a normal oxygen and a lowglucose condition.

[0021]FIG. 4c a graph showing the change in lactic acid concentrationwith incubation time under a normal oxygen and a low glucose condition.

[0022]FIG. 5 is a graph showing cell viability of HepG2 treated withvarious antibiotics.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The method for increasing survival rate of cells in animal cellculture under hypoxia condition comprises the steps of addingantibiotics of quinolones, quinones, aminoglycosides or chloramphenicolin a concentration of 0.1 to 1000 μg/ml to a culture medium andculturing the animal cells. The antibiotics includs quinolones, quinonesand aminoglycosides, but are not intended to be limited to, where thequinolone antibiotics include levofloxacin, ofloxacln and ciprofloxacin;quinone antibiotics include tetracycline, minocycline, doxycycline, andoxytetracycline; and, aminoglycoside antibiotics include geneticin,neomycin and gentamycin.

[0024] A variety of animal cells are available, which include humanhepatoma cell line HepG2 (ATCC HB 8065), human liver cell line Changliver (ATCC CCL 13), murine neuronal cell line C6 (ATCC CCL 107), humanneuroblastoma cell line BE2 cells (ATCC TIB 182), lymphoma cell line5F12AD3 (ATCC HB 8209) or bovine aortic endothelial cells (BAE). As theculture medium, it is preferred that a Minimal Essential Mediumsupplemented with 70-130 unit/ml penicillin, 90-110 μg/ml streptomycin,0.8 to 1.5 g/l glucose, 1.5 to 3 g/l sodium bicarbonate, and 8-12% (v/v)fetal calf serum is for HepG2; Dulbecco's Modified Medium supplementedwith 8-12% (v/v) inactivated fetal calf serum for BE2; Iscove's ModifiedDulbecco's Medium supplemented with 8-12% (v/v) fetal calf serum for5F12AD3.

[0025] The present inventors cultured animal cells under normalcondition and observed the changes occurred in the cells afterdiscontinuing the supply of oxygen and glucose, which revealed that thecells were died without utilization of lactic acid together withdepletion of glucose when oxygen was depleted in the cells. Further,when oxygen supply was resumed immediately after depletion of glucose,cells could survive as long as lactic acid was used up, but finallycells died with exhaustion of lactic acid.

[0026] Analyses of various test groups of cells under a condition ofoxygen and glucose depletion have shown that the groups of cells withoutantibiotic treatment underwent typical apoptosis, whereas, the groups ofcells treated with said antibiotics did not undergo apoptosis for acertain period of time. These results imply that the said antibioticsinhibit apoptosis occurred in cells with ischemic injury which lacks anadequate supply of oxygen and glucose. Additional experimentsdemonstrated that antibiotics somehow affect the expression of bcl-2protein which is known to be an inhibitor of apoptosis in cells withischemic injury.

[0027] The present invention is further illustrated in the followingexamples, which should not be taken to limit the scope of the invention.

EXAMPLE 1 Cell Viability Under Various Oxygen Conditions

[0028] HepG2 cells (human hepatoma cell line, ATCC HB 8065, 1×10⁶cells/60 mm culture dish) were grown in a Minimal Essential Mediumsupplemented with 100 unit/ml penicillin, 100 μg/ml streptomycin, 1 g/lglucose, 2.2 g/l sodium bicarbonate, and 10% (w/v) fetal calf serum for2 days, followed by feeding with the same medium and incubating under anenvironment of 1, 2, or 5% (v/v) oxygen, respectively. Numbers of viablecells with time were determined by trypan blue exclusion assay usinghemocytometer after 10-15 minutes of incubation of 1:1 (v/v) mixture of0.4% (w/v) trypan blue and cell suspension. Cell viability with time wasrepresented in the ratio of viable cell number to cell number justbefore the incubation condition was changed to a low oxygen condition(see: FIG. 1). FIG. 1 is a graph showing cell viability under variousoxygen conditions, where () indicates 1% (v/v), (▾) indicates 2% (v/v),(▪) indicates 5% (v/v), and (♦) indicates 21% (v/v) oxygen,respectively. As shown in FIG. 1, it was clearly demonstrated that HepG2cells were viable in a minimal medium containing low concentration ofglucose under an environment over 5% (v/v) oxygen, whereas, the cellsdied under an environment of less than 2% (v/v) oxygen.

[0029] Accordingly, a low oxygen condition was set at 1% (v/v) oxygen inthe following examples.

EXAMPLE 2 Dependency of Cell Viability on Geneicin Concentration

[0030] Dependency of cell viability on geneticin concentration wasdetermined under a low glucose (1 g/L) or a high glucose (4.5 g/L)condition, as well as under a low oxygen (1%, v/v) or normal oxygencondition.

EXAMPLE 2-1 Cell Viability Under a Low Oxygen (Hypoxic) and a LowGlucose (Hypoglycemic) Condition

[0031] HepG2 cells were plated in 60 mm culture dishes at a density of5×10⁵ cells per dish under a condition of 1 g/L glucose and 1% (v/v)oxygen, and then, cell viability, changes in pH and changes in glucoseconcentration were determined with time after adding 10 μg/ml geneticinor without addition (see: FIGS. 2a, 2 b and 2 c). FIG. 2a shows HepG2cell viability with incubation time, 2 b shows change in glucoseconcentration with incubation time, and 2 c shows change in pH withincubation time, where () indicates without addition and (o) indicatesaddition of 10 μg/ml geneticin. As shown in FIGS. 2a to 2 c, geneticinmaintained cell viability even after glucose was used up under a hypoxicand hypoglycemic condition.

Example 2-2 Cell Viability Under a Low Oxygen (Hypoxic) and a HighGlucose Condition

[0032] HepG2 cells were grown under the same condition described inExample 2-1 except 4.5 g/L glucose and 1% (v/v) oxygen, and then, cellviability, changes in pH and changes in glucose concentration weredetermined with time after treatment with 10 μ/g/ml geneticin or withouttreatment, respectively (see: FIGS. 3a, 3 b and 3 c). FIG. 3a showsHepG2 cell viability with incubation time, 3 b shows changes in glucoseconcentration with incubation time, and 3 c shows changes in pH withincubation time, where () indicates without treatment and (o) indicatestreatment with 10 μg/ml geneticin. As shown in FIGS. 3a to 3 c, it wasclearly demonstrated that geneticin maintained cell viability under ahypoxic and high glucose condition in a similar manner under a hypoxicand hypoglycemic condition.

Example 2-3 Cell Viability Under a Normal Oxygen (Normoxic) and a LowGlucose (Hypoglycemic) Condition

[0033] HepG2 cells were grown in the same manner as in Example 2-1except for 1 g/L glucose and 21% (v/v) oxygen, and then, cell viability,change in glucose concentration and change in lactic acid concentrationwere determined with time after adding 10 μg/ml geneticin or withoutaddition (see: FIGS. 4a, 4 b and 4 c). FIG. 4a shows HepG2 cellviability with incubation time, 4 b shows change in glucoseconcentration with incubation time, and 4 c shows change in lactic acidconcentration with incubation time, where () indicates withouttreatment and (▪) indicates treatment with 10 μg/ml geneticin. As shownin FIGS. 4a to 4 c, under normoxic condition, cells survived whileconsuming accumulated lactic acid after depletion of glucose and cellsdied with exhaustion of lactic acid, whereas, cells treated withgeneticin were viable without being affected by depletion of lacticacid.

EXAMPLE 3 Effects of Various Antibiotics on Cell Viability

[0034] In order to screen antibiotics which have similar effect togeneticin but have different chemical structure, HepG2 (Human hepatomacell line, ATCC HB 8065) cells were grown under the same conditiondescribed in Example 1, followed by replacing the medium with freshmedium proper for test conditions described below, and then, cellviabilities under various conditions were compared after 2 days ofincubation. Test groups were divided as follows depending on testconditions: test group A with 21% (v/v) oxygen and 4.5/L glucose, testgroup B with 21% (v/v) oxygen and 1 g/L glucose, test group C with 1%(v/v) oxygen and 4.5/L glucose, tesy group D with 1% (v/v) oxygen and 1g/L glucose, test group E with aminoglycoside antibiotic of geneticin(10 μg/ml) treated test group D, test group F with quinolone antibioticof ofloxacin (10 μg/ml) treated test group D, and test group G withquinone antibiotic of doxycycline (0.1 μg/ml) treated test group D (see:FIG. 5)

[0035]FIG. 5 is a graph showing the comparison of effects of variousantibiotics on the cell viability. As shown in FIG. 5, it has been foundthat: cell viability of test group D was low compared to test groups A,B and C, and cell viability of test group D can be recovered bytreatment of various antibiotics.

EXAMPLE 4 Screening of Antibiotics Exerting Positive Effects on CellViability

[0036] Based on the results obtained in Example 3 above, it was foundthat antibiotics of quinolones and quinones as well as amlnoglycosidescan enhance cell viability under hypoxia condition. In order to examinewhether antibiotics with other structures than aminoglycosideantibiotics, can also enhance cell viability under a hypoxic condition,analyses were performed as followings: i.e., after analysis ofantibiotics such as geneticin, neomycin, gentamycin, tetracycline,minocycline, oxytetracycline, doxycycline, chloramphenicol,levofloxacin, ofloxacin, cidrofloxacin, ampicillin, amoxicillin,cephalosporin, erythromycin, sulfadiazine, cyclohexamide,5-fluorouracil, puromycin and trimetazidine in accordance with theprocedure described in Examples 2-1 and 2-2, antibiotics which showedenhancement of cell viability under hypoxic condition were selected andtheir effective concentrations were determined, respectively (see: Table1). TABLE 1 Antibiotics exerting enhancement effects on cell viabilityand their effective concentration Antibiotics Concentration(μg/ml)geneticin 10-100 neomycin 1000 gentamicin 100-1000 tetracycline 0.1-10  minocycline 0.1-10   doxycycline 0.1-10   oxytetracycline 0.1-10  chloramphenicol 1-10 levofloxacin 10-100 ofloxacin 10-100 ciprofloxacin1-10

[0037] Effective concentration ranges in Table 1 represent theconcentration ranges of antibiotics exerting enhancement effects onHepG2 cell viability under 1% (v/v) oxygen condition. As shown in Table1 above, among the antibiotics known to act on 30S subunit of ribosomein E. coli, neomycin and gentamycin other than geneticin were effectiveamong aminoglycoside antibiotics. Also, among the antibiotics known toact on 30S subunit of ribosome in E. coli, a ouinone antibiotic oftetracycline was effective at very low concentration range of 0.1-10μg/ml, and tetracycline derivatives such as minocycline, oxytetracyclineand doxycycline were effective at the same range of low concentration.Meanwhile, among the antibiotics known to act on 50S subunit of ribosomein E. coli, an aromatic antibiotic of chloramphenicol was effective, buta macrolide antibiotic of erythromycin was not effective. Amongquinolone antibiotics known to act on DNA gyrase, all analyzedcompounds, levofloxacin, ofloxacin, and ciprofloxacin were effective.However, antibiotics known to inhibit synthesis of cell wall ofmicroorganisms, such as ampicillin, amoxillin, and cephalosporin did notshow enhancement effect on cell viability. Antibiotics such as asulfadiazine which is known to inhibit dihydropteroate synthetase in thefolic acid metabolism, a cyclohexamide inhibiting protein synthesis ineukaryotes, a 5-fluorouracil blocking DNA synthesis by competing withuracil, and puromycin inhibiting protein synthesis did not show anyeffect on cell viability. Based on these results, it has beendemonstrated that there is no significant relations between the abilityof antibiotics to enhance cell viability under hypoxic condition and theaction mechanism of antibiotics or the chemical structure ofantibiotics. Although efficacy of antibiotics to maintain cell viabilityunder hypoxic condition varies, effective concentration range ofantibiotics on enhancement of viability of human hepatoma cell line wasabout 0.1 to 1000 μg/ml. Meanwhile, trimetazidine which is known toenhance cell viability by increasing utilization of glucose under ahypoxic condition did nor show any positive result in the presentinvention.

[0038] As clearly illustrated and demonstrated above, the inventionprovides a method for increasing survival rate of cells in animal cellculture under hypoxia condition, which comprises the steps of addingantibiotics of quinolones, quinones, aminoglycosides or chloramphenicolin a concentration of 0.1 to 1000 μg/ml to a culture medium andculturing the animal cells. The invented method can be practicallyapplied to a mass production of recombinant protein and a high-densityanimal cell culture.

[0039] Various modifications of the invention in addition to those shownand described herein will be apparent to those skilled in the art fromthe foregoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

What is claimed is:
 1. A method for increasing survival rate of cells inanimal cell culture under hypoxia condition, which comprises the stepsof adding antibiotics of quinolones, quinones, aminoglycosides orchloramphenicol in a concentration of 0.1 to 1000 μg/ml to a culturemedium and culturing animal cells.
 2. The method for increasing survivalrate of cells in animal cell culture under hypoxia condition of claim 1,wherein the quinolone antibiotics are levofloxacin, ofloxacin andciprofloxacin.
 3. The method for increasing survival rate of cells inanimal cell culture under hypoxia condition of claim 1, wherein thequinone antibiotics are tetracycline, minocycline, doxycycline andoxytetracycline.
 4. The method for increasing survival rate of cells inanimal cell culture under hypoxia condition of claim 1, wherein theaminoglycoside antibiotics are geneticin, neomycin and gentamycin. 5.The method for increasing survival rate of cells in animal cell cultureunder hypoxia condition of claim 1, wherein the animal cells are humanhepatoma cell line HepG2 (ATCC HB 8065), human liver cell line Changliver (ATCC CCL 13), murine neuronal cell line C6 (ATCC CCL 107), humanneuroblastoma cell line BE2 cells (ATCC TIB 182), lymphoma cell line5F12AD3(ATCC HB 8209) and bovine aortic endothelial cells (BAE).